LOCUS       sequence-395517-        6939 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses CovKCR2_mCherry fusion in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6939)
  AUTHORS   Govorunova EG, Sineshchekov OA, Brown LS, Bondar AN, Spudich JL
  TITLE     Structural Foundations of Potassium Selectivity in 
            Channelrhodopsins.
  JOURNAL   mBio. 2022 Dec 20;13(6):e0303922. doi: 10.1128/mbio.03039-22. Epub 
            2022 Nov 22.
  PUBMED    36413022
REFERENCE   2  (bases 1 to 6939)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "mBio."; 
            date: "2022-12-20"; volume: "13(6):e0303922. doi"; pages: " 
            10.1128/mbio.03039-22. Epub 2022 Nov 22"
COMMENT     Sequence Label: CovKCR2_mCherry-pcDNA3.1
FEATURES             Location/Qualifiers
     source          1..6939
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1700..2407
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(1842..1860)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2110..2129
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2437..2478
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2488..2505
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2528..2545)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2534..2758
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2804..3232
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2891..2910)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3101..3122
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3241..3261)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3246..3575
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3426..3561
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3488..3507
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3642..4436
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3696..3715)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4306..4325
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4612..4733
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4649..4668)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4703..4722
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4782..4798)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4782..4798)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4795..4817)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4806..4822
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4830..4836,4837..4854,4855..4860))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4875..4896
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5013..5030)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5184..5772)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5264..5283)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5943..6734,6735..6803))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6566..6585
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6804..6908)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagt
      901 taagcttggt accgagctcg gatccatggg cgccatcgac caggatggca gcctggtgtt
      961 caagcagctg tttgacgtga actgcttcac cgccgtgttt cagaccctga catgtatgct
     1021 gctgctgtgg tccgtgatca ggggccgcgg catcaagggc agaccttatg gaccagaagg
     1081 accacctgag tctggcttca tcggcgtgat cgtgtgcatg ttcagctcct ttctgtacgt
     1141 gtgcaagtct atcgattggg gcaataaggg cacagccaac ctgtccttta ataagtactg
     1201 gtattctgac tacctggtga cctgcccact gatcgtgctg gacctgtgct tcacatttga
     1261 gatccactac aagctggtgt atttcgtgtc tacctttatc acactgctgt tcgccgtgct
     1321 gagctttacc gaggatgcac ctgcaagata ctggcacttc ggcatcggca gcgccttctt
     1381 tgccgtgttc ttttattcca tcgccaagat cctgatgcag cggtggagaa cagtgccaga
     1441 gcccggcaga atccccatgt tcgtgggcct gctgatcttc tttggcatgt ggcccgtgtt
     1501 ccctgtgctg tggatctttt cttgggtggg cagcggctac atcaccatgc accagtacta
     1561 tatcatccac tgcttcctgg acatcacctg taagacattc ttttcttttg cctgcctgct
     1621 gtatagggtg cgcgtggagg agtggcagtg gcaggccgag gtgtgcatcg atccccagcg
     1681 gagatcccag gcggccgccg tgagcaaggg cgaggaggat aacatggcca tcatcaagga
     1741 gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt tcgagatcga
     1801 gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa
     1861 gggtggcccc ctgcccttcg cctgggacat cctgtcccct cagttcatgt acggctccaa
     1921 ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct tccccgaggg
     1981 cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga
     2041 ctcctccctg caggacggcg agttcatcta caaggtgaag ctgcgcggca ccaacttccc
     2101 ctccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcct ccgagcggat
     2161 gtaccccgag gacggcgccc tgaagggcga gatcaagcag aggctgaagc tgaaggacgg
     2221 cggccactac gacgctgagg tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc
     2281 cggcgcctac aacgtcaaca tcaagttgga catcacctcc cacaacgagg actacaccat
     2341 cgtggaacag tacgaacgcg ccgagggccg ccactccacc ggcggcatgg acgagctgta
     2401 caagtaacat atgctctaga gggcccgcgg ttcgaaggta agcctatccc taaccctctc
     2461 ctcggtctcg attctacgcg taccggtcat catcaccatc accattgagt ttaaacccgc
     2521 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     2581 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     2641 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     2701 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     2761 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     2821 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     2881 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     2941 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     3001 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     3061 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     3121 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     3181 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt
     3241 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     3301 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     3361 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     3421 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     3481 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt
     3541 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca
     3601 ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat
     3661 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac
     3721 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc
     3781 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc
     3841 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag
     3901 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc
     3961 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg
     4021 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc
     4081 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc
     4141 cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga
     4201 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca
     4261 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg
     4321 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg
     4381 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg
     4441 gactctgggg ttcgcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc
     4501 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
     4561 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt
     4621 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     4681 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     4741 tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg
     4801 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     4861 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     4921 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     4981 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     5041 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     5101 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     5161 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     5221 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     5281 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     5341 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     5401 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     5461 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     5521 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     5581 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     5641 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     5701 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     5761 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     5821 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     5881 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     5941 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     6001 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     6061 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     6121 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     6181 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     6241 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     6301 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     6361 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     6421 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     6481 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     6541 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     6601 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     6661 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     6721 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     6781 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     6841 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     6901 gttccgcgca catttccccg aaaagtgcca cctgacgtc
//
